granule-associated pruritogenic media-tor from mast cells, histamine, weremarkedly inhibited by PAC-14028,whereas those by serotonin or throm-boxane analog, U-46619, were notaffected. Partial attenuation of hista-mine-mediated itch response might re-flect the partial agonistic effect ofhistamine on TRPV1 activation (Shimet al., 2007), and the lack of antipruriticefficacy on the scratching behaviorsevoked by serotonin or U-46619 wasanticipated as these pruritogens workthrough G-protein-coupled receptors,including 5-HT receptors and TP recep-tor (Andoh et al., 2007; Imamachi et al.,2009), that have little cross talk withTRPV1-mediated pathways. Partial inhi-bition of PAC-14028 on compound 48/80-evoked itch may be explained by thepresence of TRPV1-independent itchsignals from serotonin or other prurito-genic mediators in mast cell granules,despite histamine- and tryptase-mediated(PAR-2 mediated) itch responses couldbe suppressed by TRPV1 blockade.

In conclusion, we found that TRPV1antagonist, PAC-14028, can suppressscratching behaviors associated withAD-like symptoms through the inhibitionof TRPV1 activation, of which expressionand phosphorylation increase in AD-likeskin lesion. Particularly, experimentswith diverse pruritogens revealed thatPAC-14028 could manifest antipruriticeffects on TRPV1-, PAR-2-, and hista-mine-mediated scratching behaviors.Considering the frequent failure ofconventional antipruritic therapy (i.e.,

antihistamines) in the management ofsevere itch symptoms, we believe thatTRPV1 antagonists can be a novel anti-pruritic therapy that might satisfy theunmet medical need.

CONFLICT OF INTERESTPAC-14028 is the patented compound of AmorePacific Corporation (Patent PCT/KR07/003592:novel compounds, isomer thereof, or pharmaceu-tically acceptable salts thereof as vanilloid recep-tor antagonist, and pharmaceutical compositionscontaining the same).

ACKNOWLEDGMENTSThis work was supported by a grant from theMinistry of Knowledge Economy (Bio-Star,10031636).

Jun-Won Yun1, Jung A Seo1, Won-HeeJang1, Hyun Ju Koh1, Il-Hong Bae1,Young-Ho Park1 and Kyung-Min Lim1

1AmorePacific Corporation R&D Center,Medical Beauty Research Institute,Gyeonggi-do, Republic of KoreaE-mail: kimlim@amorepacific.com

REFERENCES

Andoh T, Nishikawa Y, Yamaguchi-Miyamoto Tet al. (2007) Thromboxane A2 induces itch-associated responses through TP receptors inthe skin in mice. J Invest Dermatol 127:2042–7

Bae IH, Yun JW, Seo JA et al. (2010) Immunohis-tological comparison of cutaneous pathologyof three representative murine atopic derma-titis models. J Dermatol Sci 59:57–60

Caughey GH (2007) Mast cell tryptases andchymases in inflammation and host defense.Immunol Rev 217:141–54

Chan LS, Robinson N, Xu L (2001) Expression ofinterleukin-4 in the epidermis of transgenic miceresults in a pruritic inflammatory skin disease:an experimental animal model to study atopicdermatitis. J Invest Dermatol 117:977–83

Costa R, Marotta DM, Manjavachi MN et al. (2008)Evidence for the role of neurogenic inflammationcomponents in trypsin-elicited scratching beha-viour in mice. Br J Pharmacol 154:1094–103

Hutter MM, Wick EC, Day AL et al. (2005)Transient receptor potential vanilloid (TRPV-1)promotes neurogenic inflammation in thepancreas via activation of the neurokinin-1receptor (NK-1R). Pancreas 30:260–5

Imamachi N, Park GH, Lee H et al. (2009) TRPV1-expressing primary afferents generate behavioralresponses to pruritogens via multiple mecha-nisms. Proc Natl Acad Sci USA 106:11330–5

Ohmura T, Hayashi T, Satoh Y et al. (2004)Involvement of substance P in scratchingbehaviour in an atopic dermatitis model.Eur J Pharmacol 491:191–4

Shim WS, Tak MH, Lee MH et al. (2007) TRPV1mediates histamine-induced itching via theactivation of phospholipase A2 and 12-lipoxygenase. J Neurosci 27:2331–7

Shimada SG, Shimada KA, Collins JG (2006)Scratching behavior in mice induced bythe proteinase-activated receptor-2 agonist,SLIGRL-NH2. Eur J Pharmacol 530:281–3

Steinhoff M, Bienenstock J, Schmelz M et al.(2006) Neurophysiological, neuroimmunolo-gical, and neuroendocrine basis of pruritus.J Invest Dermatol 126:1705–18

Steinhoff M, Griffiths CEM, Church MK et al.(2004) Inflammation. In: Rook’s Textbook ofDermatology (Burns T, Breathnach S, Cox N,Griffiths C, eds). Oxford: Blackwell Publish-ing, 251–318

Velazquez RA, McCarson KE, Cai Y et al. (2002)Upregulation of neurokinin-1 receptor expressionin rat spinal cord by an N-terminal metabolite ofsubstance P. Eur J Neurosci 16:229–41

Yamaoka J, Kawana S (2007) Rapid changes insubstance P signaling and neutral endopepti-dase induced by skin-scratching stimulationin mice. J Dermatol Sci 48:123–32

Yun J-W, Seo JA, Jeong YS et al. (2011) TRPV1antagonist can suppress the atopic dermatitis-like symptoms by accelerating skin barrierrecovery. J Dermatol Sci 62:8–15

Global Analysis of BRAFV600E Target Genes in HumanMelanocytes Identifies Matrix Metalloproteinase-1 as aCritical Mediator of Melanoma GrowthJournal of Investigative Dermatology (2011) 131, 1579–1583; doi:10.1038/jid.2011.65; published online 31 March 2011

TO THE EDITORBRAF kinase has been found to bemutationally activated in up to 70% of

benign nevi and melanomas (Davieset al., 2002). It has been implicated as acritical mediator of melanoma devel-

opment, with the V600E-activating mu-tation representing the most commonlymutated form of BRAF in nevi andmelanomas (Pollock et al., 2003).Despite strong evidence implicatingBRAF kinase as a bona-fide oncogene

Abbreviations: EGFR, epidermal growth factor receptor; HPM, human primary melanocyte; MMP-1,matrix metalloproteinase-1; siRNA, small interfering RNA

www.jidonline.org 1579

B Ryu et al.BRAF Activates Melanocyte MMP-1

in melanoma, its precise downstreamtargets in melanocytes have not beendefined to date, and a BRAF-specificgene signature in melanomas remainsuncertain (Hoek et al., 2006).

We have introduced activatedBRAFV600E into human primary melano-cytes (HPMs) in order to assess its specificfunctions (Figure 1a, SupplementaryFigure S2 online; also see Supplementaryinformation for details of experimentalmethods). The gene expression signatureof HPMs induced by acute expression ofthe BRAFV600E was assessed in compar-ison with HPMs expressing green fluor-escent protein (Figure 1c). The completedataset is accessible as GSE13827(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13827). We found thatthe BRAFV600E signature of HPMs wascharacterized by upregulation of sev-eral growth-promoting genes andcellular motility and inflammation-associated genes (Figure 1b) with acommon network activation of cellulargrowth/proliferation and apoptosis(Supplementary Figure S1 online). Thisfinding suggests that BRAFV600E mayinduce gene signatures of biphasiccellular responses, proliferation asobserved in melanomas, and onco-gene-induced growth arrest as observed

in nevi. Detection of genes involved inproliferation, such as MMP1, AREG,CXCL5, IL-8, and EREG (Figure 1c andSupplementary Table S1 online), im-plies that the short-term cellular re-sponse by acute BRAFV600E expressionmay be HPM proliferation rather thangrowth arrest, which appears to be along-term sustained effect of BRAFV600E

(Woods et al., 1997; Michaloglou et al.,2005). Therefore, we reasoned that thecellular proliferation signal induced bythe BRAFV600E in HPMs may be reacti-vated in melanoma for tumor growth. Inorder to test this hypothesis, we soughtto further examine the functional rolesof matrix metalloproteinase-1 (MMP-1)as a BRAF effector in melanoma be-cause MMP-1 is most strongly inducedby BRAF (Supplementary Table S1 on-line) and is reported to be involved inmelanoma progression and metastasis(Blackburn et al., 2007).

In order to determine whetherMMP-1 expression is correlated withBRAFV600E expression in melanomas,melanoma cell lines and HPMs wereexamined for MMP-1 messenger RNAexpression using gene expression pro-filing as previously described (Ryuet al., 2007). We identified 25-foldincreased expression of MMP-1 mes-

senger RNA in melanoma cells posses-sing BRAFV600E compared with wildtype whereas HPMs expressed similartranscript levels with BRAF wild-typemelanoma cell (Figure 2a), suggestingthat increased BRAF kinase activity maybe associated with elevated MMP-1expression in melanomas. We alsofound that melanocytes expressingBRAFV600E have increased levels ofsecreted MMP-1 protein (Figure 2b)and collagenase activity (Figure 2c)versus HPM controls, suggesting thatthe activated BRAF can induce bothMMP-1 protein expression and activity.In order to determine the functionalsignificance of BRAF kinase inductionof MMP-1 in human melanomas, weassessed the effect of MMP-1 genesilencing on the proliferative functionsof BRAF kinase. MMP-1 messengerRNA and protein levels were efficientlyreduced in melanomas possessingeither wild-type BRAF (WM852) ormutant BRAFV600E (WM793) using tar-geted MMP-1 small interfering RNA(siRNA) (Figure 2d and e). Cellularproliferation was assessed in both BRAFwild-type and mutant melanomas fol-lowing MMP-1 silencing by siRNA(Figure 2f) and a neutralizing MMP-1antibody (data not shown). Significant

BR

AF

V60

0E

BR

AF

-dea

d

GF

P

1205

Lu

WM

938B

BRAF

p-MEK1/2

GF

P-1

GF

P-2

BR

AF

V60

0E-1

BR

AF

V60

0E-2

MEK1/2

GO biological process

Cell-cycleprogression (11)

Acuteinflammation (3)

Inflammatoryresponse (7)

Cellproliferation (9)

Locomotorybehavior (5)

Homeostaticprocess (7)

Chemotaxis (2)

Response tostimulus (19)

MMP1: metalloendopeptidase activitySERPINB2: serine-type endopeptidaseAREG: growth factor activitySCG5: intracellular protein transportIL8: chemokine activityTFPI2: serine-type endopeptidase inhibitior

CXCL5: cytokine activityEREG: EGFR bindingBRAF: MAPKKK activityAGPAT9: regulation of TOR signaling pathwayRAP1A: ras GTPasa binding

DKK1: multicellular organismal developmentAPC: beta-catenin binding

CLIP1: microtubule binding

RB1CC1: kinase activityDNAJB14: heat shock protein binding

ZNF292: DNA bindingZNF277: ---MYH10: actin bindingDENND1B: ---EMCN: carbohydrate bindingUBXN4: protein bindingSNRPN: RNA binding

LRRC39: protein bindingDLEU2: ---PSIP1: DNA binding

CCL2: CCR2 chemokine receptor binding

STC1: hormone activity

IL24: cytokine activityBEX1: ---

Figure 1. A proliferative gene signature induced by oncogenic BRAF in human primary melanocyte (HPM) and gene ontology (GO) annotation analysis.

(a) Activation of mitogen-activated protein kinase signal transduction pathway by acute expression of BRAFV600E in HPM. (b) A pie chart of the GO

annotation analysis. Eighty-two annotated genes from 137 probe sets that were identified as greater than threefold differentially expressed genes were

analyzed. P-value o0.005 was used for identification of the biological processes that may be regulated by BRAF downstream effectors. Numbers shown

in parentheses indicate number of genes classified as the suggested categories. (c) Heat map presentation of the downstream effector gene signature

induced by BRAFV600E. Top 15 up- and downregulated genes are shown. BRAFV600E; HPM expressing mutant BRAFV600E; GFP, HPM expressing

green fluorescent protein.

1580 Journal of Investigative Dermatology (2011), Volume 131

B Ryu et al.BRAF Activates Melanocyte MMP-1

inhibition of proliferation was seen inboth BRAF wild-type and mutant mel-anoma cells following MMP-1 knock-down; however, whereas cell growthwas inhibited only by 17% with MMP-1

siRNA versus control siRNA in BRAFwild-type melanomas, growth inhibi-tion by MMP-1 siRNA in the BRAFmutant melanoma cells was signifi-cantly more effective, at 80% inhibi-

tion, despite comparable gene silencing(Figure 2f). We therefore conclude thatBRAFV600E may promote cellulargrowth in melanomas through activatedexpression of MMP-1. It should be

MM

P-1

mR

NA

leve

l(h

ybrid

izat

ion

unit

×1,0

00) 14

8

0R

elat

ive

mR

NA

leve

l 1.4

1.0

0.6

0.0

Cel

l pro

lifer

atio

n(%

of c

ontr

ol)

Scramble

WM852 WM793

siMMP-1 Scramble siMMP-1

120

100

80

60

40

0

20

Rel

ativ

e M

MP

-1 c

onc.

20

10

0

Rel

ativ

e A

RE

G c

onc. 100

80

60

40

20

0Rel

ativ

e A

RE

G c

onc.

80

40

0Control

Control siMMP-1

WM852 WM793

Scramble Control siMMP-1 ScrambleBRAF

Scramble ScramblesiMMP-1 siMMP-1

WM852 WM793

Control siMMP-1 Scramble

WM852

Control siMMP-1 Scramble

WM793

HPM Control BRAF

******

******

*

***

***

*** ***

*

Control BRAF0

5

9

Rel

ativ

e M

MP

-1 a

ctiv

ity(×

1,00

0)

Rel

ativ

e M

MP

-1 c

onc.

0

20

40

WM852 WM793

Figure 2. Activated BRAF promotes melanoma cell growth by matrix metalloproteinase-1 (MMP-1). (a) Relative MMP-1 messenger RNA levels in human

primary melanocytes (HPMs) and melanoma cells expressing wild-type (WM852) or mutant BRAF (WM793). (b) Relative levels of secreted MMP-1 in

conditioned media obtained from HPMs expressing green fluorescent protein (GFP) or BRAFV600E at 72 h following lentiviral infection. (c) Relative MMP-1

collagenase activity in conditioned media obtained from HPMs expressing GFP or BRAFV600E at 72 h following lentiviral infection. (d) Quantitative real-time

RT-PCR (qRT-PCR) analysis of MMP-1 expression following gene silencing by small interfering (siRNA) in melanomas possessing either wild-type (WM852) or

mutant BRAFV600E (WM793). (e) Relative MMP-1 concentration in cell culture media following MMP-1 gene silencing in melanomas possessing wild-type

(WM852) and BRAFV600E (WM793) cells. (f) 3H-thymidine cell proliferation assay of melanomas possessing wild-type (WM852) and BRAFV600E (WM793)

following MMP-1 gene silencing. (g) Relative expression of activated amphiregulin (AREG) in conditioned media from HPMs expressing GFP or BRAFV600E.

(h) Relative expression of activated AREG in melanomas expressing wild-type (WM852) or mutant BRAF (WM793) following MMP-1 gene silencing.

Columns: mean of three individual experiments done in triplicate; bars, SD. *Po0.05, **Po0.01, ***Po0.001, compared with GFP control in b, c, g, and

compared with siRNA control (Scramble) in the d–f, h.

www.jidonline.org 1581

B Ryu et al.BRAF Activates Melanocyte MMP-1

noted that MMP-1 silencing by RNAinterference was previously shown toaffect only metastasis but not tumorgrowth in a melanoma cell line(VMM12) (Blackburn et al., 2007).However, Blackburn et al. (2009) alsoreported in a recent study that stableoverexpression of MMP-1 in Bowersmelanoma cells promotes xenografttumor growth. These data suggest vari-able effects of MMP-1 in melanoma celllines, which are likely attributable tothe molecular heterogeneity of melano-mas. It is likely that WM792 andBowers melanoma cells depend onBRAF/MMP-1-mediated cellular path-ways for tumor growth, which may notbe the case for VMM12 melanomacells. As we were able to show thatMMP-1 promotes growth in melanomacells expressing BRAFV600E, we soughtto clarify the functional targets forMMP-1 in this setting. As amphiregulin(AREG), a ligand for the epidermalgrowth factor receptor (EGFR), was alsofound to be significantly induced byBRAFV600E in HPMs (Figure 1c andSupplementary Table S1 online) and issynthesized as a precursor protein thatis released from the plasma membraneby metalloproteinases (Zhang et al.,2004; Lu et al., 2009), we sought toevaluate whether HPMs expressingBRAFV600E expressed elevated levels ofactivated AREG. We found 4100-foldexpression of activated AREG in HPMsexpressing BRAFV600E versus controls(Figure 2g). In order to determinewhether induction of activated AREGin HPMs expressing BRAFV600E was dueto cleavage by activated MMP-1, weevaluated the effect of silencing MMP-1on expression of activated AREG inmelanoma cells expressing BRAFV600E

versus wild-type BRAF-expressing mel-anoma cells. We found that silencing ofMMP-1 led to a significant reduction inlevels of cleaved AREG in BRAFV600E

melanoma cells, but no signi-ficant change in expression in theBRAF wild-type melanoma cells(Figure 2h).

Although previous studies suggestedthat BRAF kinase activity promotesexpression of MMP-1 in melanoma(Huntington et al., 2004) and thatMMP-1 promotes melanoma progres-sion (Blackburn et al., 2007), these

authors conclude that induction ofMMP-1 in melanoma is specificallyimportant for melanoma progressionand metastasis through degradationfunctions on interstitial collagens. Herewe show that MMP-1 is a criticalmediator of the growth-promoting func-tions of BRAF kinase in melanomacells, which is consistent with a pro-liferative role for BRAFV600E in thedevelopment of melanomas. Indeed,recent studies have suggested an addi-tional important role for MMPs inactivating latent growth factors thatmay be critical to the effects of MMP-1seen in our studies. Notably, MMP-1has been implicated in activating breastcancer and melanoma cell growththrough proteolytic activation of thecell surface receptor PAR1 (Boire et al.,2005; Blackburn et al., 2009). Togetherwith the report that EGFR is highlyexpressed in vertical-growth-phase pri-mary (89%) and metastatic melanoma(80%) (Rodeck et al., 1991), the datapresented in this study demonstratingthe growth-promoting function ofMMP-1 in human melanomas suggestthat BRAFV600E-induced activation ofan autocrine feedback loop (MMP-1/AREG/EGFR/RAS/BRAF) may have acritical role in melanoma growth andmetastasis. It is also possible that tumorcell-induced AREG expression and ac-tivation may affect the growth ofneighboring endothelial and stromalcells. This may further promote tumorcell metastasis by modulating the tu-mor-specific microenvironment. Takentogether, this feedback loop could bean important player for melanomaprogression and a molecular target formelanoma therapy. Further studies arecurrently ongoing to test this hypothesisusing EGFR inhibitors in our experi-mental model.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSWe thank B Vogelstein, P Cole, and members ofthe Alani lab for their critical review of thismanuscript and helpful discussions. We thank MHerlyn, X Yu, and G Robertson for providingcritical reagents. This work was supported byNational Cancer Institute grants CA107017 (RA)and CA113779 (BR), the Flight Attendant MedicalResearch Institute (RA), The American SkinAssociation (RA, AD), The Murren Family

Foundation, and The Henry and Elaine KaufmanFoundation.

Byungwoo Ryu1,5, Whei F. Moriarty1,2,Megan J. Stine1,2, Amena DeLuca1,Dave S. Kim1, Alan K. Meeker1,3,Landon D. Grills1,Rebecca A. Switzer1, Mark S. Eller4

and Rhoda M. Alani1,2,4

1The Sidney Kimmel Comprehensive CancerCenter, Johns Hopkins University School ofMedicine, Baltimore, Maryland, USA;2Program in Cellular and Molecular Medicine,Johns Hopkins University School of Medicine,Baltimore, Maryland, USA; 3Department ofPathology, Johns Hopkins University School ofMedicine, Baltimore, Maryland, USA and4Department of Dermatology, BostonUniversity School of Medicine, Boston,Massachusetts, USAE-mail: alani@bu.edu or bryu@nvcancer.org5Current address: Department of CancerBiology, Nevada Cancer Institute,One Breakthrough Way, Las Vegas,Nevada, USA.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the onlineversion of the paper at http://www.nature.com/jid

REFERENCES

Blackburn JS, Liu I, Coon CI et al. (2009) A matrixmetalloproteinase-1/protease activated recep-tor-1 signaling axis promotes melanoma inva-sion and metastasis. Oncogene 28:4237–48

Blackburn JS, Rhodes CH, Coon CI et al. (2007)RNA interference inhibition of matrixmetalloproteinase-1 prevents melanomametastasis by reducing tumor collagenaseactivity and angiogenesis. Cancer Res67:10849–58

Boire A, Covic L, Agarwal A et al. (2005) PAR1 is amatrix metalloprotease-1 receptor that pro-motes invasion and tumorigenesis of breastcancer cells. Cell 120:303–13

Davies H, Bignell GR, Cox C et al. (2002)Mutations of the BRAF gene in humancancer. Nature 417:949–54

Hoek KS, Schlegel NC, Brafford P et al. (2006)Metastatic potential of melanomas defined byspecific gene expression profiles with no BRAFsignature. Pigment Cell Res 19:290–302

Huntington JT, Shields JM, Der CJ et al. (2004)Overexpression of collagenase 1 (MMP-1) ismediated by the ERK pathway in invasivemelanoma cells: role of BRAF mutation andfibroblast growth factor signaling. J BiolChem 279:33168–76

Lu X, Wang Q, Hu G et al. (2009) ADAMTS1and MMP1 proteolytically engage EGF-likeligands in an osteolytic signaling cascade forbone metastasis. Genes Dev 23:1882–94

Michaloglou C, Vredeveld LC, Soengas MS et al.(2005) BRAFE600-associated senescence-likecell cycle arrest of human naevi. Nature436:720–4

1582 Journal of Investigative Dermatology (2011), Volume 131

B Ryu et al.BRAF Activates Melanocyte MMP-1

Pollock PM, Harper UL, Hansen KS et al. (2003)High frequency of BRAF mutations in nevi.Nat Genet 33:19–20

Rodeck U, Melber K, Kath R et al. (1991)Constitutive expression of multiple growthfactor genes by melanoma cells but not normalmelanocytes. J Invest Dermatol 97:20–6

Ryu B, Kim DS, Deluca AM et al. (2007)Comprehensive expression profiling of tumorcell lines identifies molecular signatures ofmelanoma progression. PloS One 2:e594

Woods D, Parry D, Cherwinski H et al. (1997) Raf-induced proliferation or cell cycle arrest isdetermined by the level of Raf activity with

arrest mediated by p21Cip1. Mol Cell Biol17:5598–611

Zhang Q, Thomas SM, Xi S et al. (2004) SRCfamily kinases mediate epidermal growthfactor receptor ligand cleavage, proliferation,and invasion of head and neck cancer cells.Cancer Res 64:6166–73

UVA Induces Lesions Resembling Seborrheic Keratosesin Mice with Keratinocyte-Specific PTEN DownregulationJournal of Investigative Dermatology (2011) 131, 1583–1586; doi:10.1038/jid.2011.33; published online 10 March 2011

TO THE EDITORSeborrheic keratoses (SKs) are amongthe most common benign tumors inhumans, occurring in 80–100% of peopleover 50 years of age (Yeatman et al.,1997; Kwon et al., 2003). Although notlife-threatening, SKs may become irri-

tated and itchy, are often unattractive anddisfiguring, and may have a significantlynegative psychological impact as dailyreminders of aging. Several recent studiesof the pathogenesis of SK have identi-fied the importance of somatic activatingmutations of fibroblast growth factor

receptor 3 (FGFR3) (Logie et al., 2005;Hafner et al., 2006a, b, 2007a, b, c)and oncogenic mutations of the p110asubunit (PIK3CA) of phosphatidyl-inositol 3-kinase (PI3K) in an indepen-dent distribution (Hafner et al., 2007d,2008).

100

+/+/Sham+/+/UVA+/–/Sham

80

Tum

or-f

ree

mic

e (%

)

60

4040

+/–/UVA

5030Weeks

20

PTEN

PTEN

β-Actin

+/+ +/–+/– SK

0 0

4/6 2/6

SCC

Sham

UVA

Figure 1. PTEN (phosphatase and tensin homolog deleted on chromosome 10) hemizygosity is a predisposing factor for skin tumorigenesis following UVA

irradiation. (a) Immunoblot analysis of PTEN protein levels in the epidermis from K14Cre;Ptenþ /þ (þ /þ ) and K14Cre;Ptenfl/þ (þ /�) mice. (b) Percentage of

tumor-free mice (n¼ 15). (c) Incidence of seborrheic keratosis (SK) and squamous cell carcinoma (SCC). (d) A mouse with an SK (indicated by a red arrow)

developing in its skin. (e–h) Hematoxylin and eosin staining of the mouse specimens with SK and SCC. (e) SK at � 2.5 magnification. (f) SK at original

magnification � 10. (g) SCC at original magnification � 10. (h) SCC at original magnification �10, with a smaller field of view. (e) Scale bar¼400 mm and

(f–h) scale bar¼100 mm. The green arrow in (h) indicates a multinucleated cell.

Abbreviations: AKT, a serine–threonine kinase, downstream of PI3K, also called protein kinase B; ERK,extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensinhomolog deleted on chromosome 10; SCC, squamous cell carcinoma; SK, seborrheic keratosis; þ /þ ,Ptenþ /þ , K14Cre, Ptenþ /þ ; þ /�, Ptenþ /�, K14Cre, Ptenfl/þ

www.jidonline.org 1583

M Ming et al.SK in UVA-PTEN Interaction in Mice